The bacterial osmoregulatory network is needed to maintain and adjust the concentration difference across the cellular membrane in response to frequent, often sudden, changes in the external osmolarity. Research in bacterial osmoregulation has been extensive and peaked at about three decades ago, when predominantly in vivo, bulk measurements and genetic manipulations identified the components of the network and demonstrated that the ability to sense and respond to external concentration changes is a key adaptation mechanism. A bacterial cell that has not adapted to an increase in external concentration will stop growing, and in the event it has adapted, will grow slower. While the scope of what can be learned with earlier employed methods has been, by and large, explored, many fundamental questions remain unanswered: for instance, how is a shock sensed, when does the recovery begin, when does it end, in what temporal order the different osmorecovery mechanisms come into play and how are the recovered cells different from those that have not experienced an osmotic challenge.
Our lab aims to answer some of these important questions using Escherichia coli as the model organism.
In order to do so, we are developing tools optimized for single-cell studies of osmoregulatory network by integrating state-of-the-art fluorescence imaging techniques, microfluidic devices and optical trapping techniques.
Single bacterial cells are observed during osmotic chalanges. A sequence of images obtained is converted to high resolution trace showing changes in cellular volume. Watch the movie of the sequence of fluorescent images shown in the middle.
The developed systems are utilized to determine and understand the cascade of events and alterations a cell will undergo during and after it experiences an osmotic change in the external environment, all of which will subsequently results in a slower rate of growth.
In addition, we are currently driving the approach further, towards concurrent, single-cell and single-molecule in vivo measurements of the interactions between several different stress response networks, such as pH homeostasis and osmoregulation, chemotaxis, or membrane voltage and osmotic regulation. The entwined nature of different stress response networks makes these types of measurements crucial for understanding the differences between the states of bacterial cells prior to and post osmotic shock recovery.
Our home built microscope and optical trapping system
Synthetic biology and biotechnology applications
Lead by our increasing capability to synthesize and insert desired genetic information into the living cell, synthetic biology has become a booming discipline with great promise. However, while we are currently efficiently assembling artificial circuits and pushing the capabilities of synthesizing DNA, one of the key limitations of successfully harnessing their signal processing and manufacturing capabilities is our lack of understanding and control, over the cross-talk between the engineered circuits and the cellular chassis hosting them.
Cellular physiology can be modulated in several different ways, including temperature, antibiotics, toxins, nutrients and osmolarity. Osmotically induced changes in growth rates represent a challange for biotechnology, where high-level excretion of substrates during biotechnological applications constantly increases medium concentration, and synthetic biology, where changes in external osmolarities adversly affect the function of cell based biosensors. We are currently exploring different approaches to overcome these limitations.
Changes in bacterial physiological traits during treatment with antibiotics
Bacterial strains resistant to antibiotics are constantly emerging. Bacterial infections seriously increase the risk of even minor surgeries, target human population across all ages, and, if untreated, lead remarkably quickly to life threatening outcomes. Biofim associated infections pose an even bigger problem. The bacteria in the biofim are several fold more resistant to antibiotics. Exactly why, is far from understood. The complexity of molecular interactions initiated by antibiotic binding the target make it extraordinary difficult to elucidate the underlying mechanism(s) of cell lysis, in particular once the biofilm has formed. We are tackling the complexity of the problem by correlating the intricate network of molecular events that lead to cell death, to the changes in cell physiology of individual cells. We are particullary interested in exploring the 'temporal function' of changes in cellular physiology during antibiotic treatment, the effects the characteristic profiles have on combinatorial drug therapies, as well as in the changes in cellular physiology during early stages of biofilm formation.
(1) Pilizota T, Shaevitz JW. Fast, multiphase volume adaptation to hyperosmotic shock by Escherichia coli. PLoS ONE 2012 Apr; 7(4): e35205
(2) Pilizota T, Shaevitz JW. Plasmolysis and cell shape depend on solute outer-membrane permeability during hyperosmotic shock in Escherichia coli (2012) Submitted. Email for preprint.
(3) Wood JM. Osmosensing by bacteria. Sci STKE 2006(357):pe43
(4) Csonka LN, Hanson AD. Prokaryotic osmoregulation: genetics and physiology. Annu. Rev. Micrbiol. 1991 45:569:606
Left: A sinlge cell exposed to an increase in external concentration caused by addition of NaCl to the media. Red marks the total cell volume and green the cytoplasmic cell volume. Middle: Cells are exposed to an increase in external media and observed during recovery. Cytoplasmic cell volume is shown. Faster then real time. Right: Cells are exposed to a very large increase in external sucrose concntration. Total cell volume is shown.