Exploring the effects of various growth conditions on gene expression of mechanosensitive ion channels in Escherichia coli

Presenter Information

Sarah C. Johnson
Hannah R. Malcolm

Faculty Sponsor

Dr. Hannah R. Malcolm

Faculty Sponsor College

College of Arts and Sciences

Faculty Sponsor Department

Chemistry

Location

SOARS Virtual Conference

Presentation Website

https://unfsoars.domains.unf.edu/2021/posters/exploring-the-effects-of-various-growth-conditions-on-gene-expression-of-mechanosensitive-ion-channels-in-escherichia-coli/

Keywords

SOARS (Conference) (2021 : University of North Florida) – Archives; SOARS (Conference) (2021 : University of North Florida) – Posters; University of North Florida -- Students -- Research – Posters; University of North Florida. Office of Undergraduate Research; University of North Florida. Graduate School; College students – Research -- Florida – Jacksonville – Posters; University of North Florida – Undergraduates -- Research – Posters; University of North Florida. Department of Chemistry -- Research – Posters; Escherichia coli -- Molecular genetics – Posters; Escherichia coli -- Gene expression -- Posters

Abstract

In rapidly changing environmental conditions, bacterial mechanosensitive ion channels are responsible for ensuring cell survival. Bacterial mechanosensitive channels gate in response to membrane tension in order to relieve intracellular pressure, prior to cell lysis. The most well-studied bacterial mechanosensitive channels include the mechanosensitive channel of large (MscL) and small (MscS) conductance from Escherichia coli (E. coli), both of which gate in response to tension. In E. coli, there are seven mechanosensitive ion channel genes: MscL, MscS, and five additional genes that are members of the MscS superfamily. Of these seven genes, six have been shown to gate directly in response to tension applied to the cellular membrane. We determine each channel’s expression in varying growth conditions like salt concentration, pH, and temperature. To determine each gene’s contribution to survival, we utilized qPCR and compared mRNA levels of each individual channel to a standard ribosomal gene, RpoB, within each growth condition. The relative expression of each channel provides insight into the specific roles that each of these channels fulfills during the survival response of the cell. Throughout our data, we see that MscL plays an active role in cell survival across various conditions. We rarely see any indication of YnaI expression, with the exception of cells cultured at lower temperatures. Apart from these channels, we observe similarities in expression levels between MscS, MscM, and YbdG as well as between MscK and YbiO across various conditions. This research shows that the expression of mechanosensitive channels fluctuates based on the growth environment.

Comments

Audio Presentation Transcript:

Hello! My name is Sarah Johnson. I am an undergraduate research assistant in Dr. Hannah R. Malcolm’s research lab here at the University of North Florida. Today, we will be exploring gene expression of the seven mechanosensitive ion channels found in E. Coli by using qPCR.

To begin, we will discuss some background information regarding bacterial mechanosensation.
An important question that we must ask is: “What is bacterial mechanosensation and what does it do?” Bacterial mechanosensation allows cells to sense their environment by detecting osmotic pressures, chemical signals, and tension. By enabling a cell to sense and react to its surroundings, bacterial mechanosensation is an essential survival tool for bacterial cells. In bacteria, mechanosensitive ion channels rescue the bacteria from cell lysis by relieving intracellular pressure in response to rapidly changing environmental conditions such as membrane tension and osmotic shifts. Bacterial mechanosensitive ion channels play a key role in a cell’s survival response. The most well studied bacterial mechanosensitive ion channels, from E. coli, are the mechanosensitive channel of large (MscL) and small (MscS) conductance. These channels gate in response to tension within the membrane. Bacterial mechanosensitive channels generally fall into one of two categories: channels homologous to McsL – or – members of the MscS superfamily.

Analysis of the E. coli genome identified 5 additional mechanosensitive genes, which include: MscK, MscM (formally known as YjeP), YbdG, YnaI, and YbiO. There is little data surrounding each of these channels and their specific functions. So, through this study, we reveal each gene’s mRNA levels, their level of expression, as compared to a standard ribosomal housekeeping gene called RpoB. We subjected cells to varying growth conditions in order to provide insight into how each of these genes specifically contributes to cell survival. The E. coli genome contains a total of 7 unique channels that are predicted to gate in response to mechanical tension. Of these 7 genes in E. coli, 6 have been shown to directly gate in response to tension. Channels like MscS within the MscS superfamily respond to other stimuli based on their external conditions as well.
Members of this superfamily contain differences among both the n and c termini in comparison to MscS.The table shown at the top right of the poster shows these 7 channels and their predicted functions.

We are interested in seeing how external conditions during growth affects the expression of each gene. In order to understand how these channels are expressed, we utilized qPCR to analyze at the mRNA levels of expression under varying growth conditions. qPCR – quantitative polymerase chain reaction – is a valuable tool when it comes to identifying gene expression.
It provides us with quick but thorough quantification of collected mRNA, which we were able to utilize to compare gene expression across varying growth conditions. In order to standardize the expression of each gene, again, we utilized RpoB, the beta subunit of the ribosomal polymerase, as the internal standard. This allowed us to compare which genes showed upregulation or downregulation in each condition.

Now let’s look into our data: starting in the yellow box, based on the growth curve of E. coli, we wanted to determine if the expression levels of these genes are consistent or vary throughout the different phases of growth. We looked at mid-log, steady state, and an overnight culture. The time points that we selected to collect mRNA from are circled in red. Error bars signify an n=3.
Compared to the internal standard, RpoB, all genes were expressed at a higher level when pulled from steady-state in comparison to levels observed during mid-log phase. At 1.0, the genes are expressed at a similar level compared to a housekeeping gene, in this case: RpoB. In both steady-state and mid-log phase, MscL is the highest expressed gene, above the expression of RpoB in its steady state and overnight culture. MscS and YbdG follow MscL by both being expressed at a level similar to RpoB at 1.0 in their steady and overnight states.

Moving over to the red box, observing gene expression at high salt, we compared gene expression at 0.5M NaCl and 0.5M KCl. In both high NaCl and high KCl, we observed that MscL is upregulated. In high NaCl, we see that MscM is slightly downregulated. In KCl, we observed that YbiO is slightly downregulated. With the high KCl environment, an interesting takeaway from this set of data is seen with our finding that MscK is not found to be highly expressed though subjected to a potassium-dense environment – it is expressed slightly more in NaCl than KCl. This is notable as MscK’s predicted function is potassium activated.

Moving down to the green box, we were also able to explore the effects of varying temperature and pH in culturing conditions. First let’s look at the pH on the right. We experimented with varying pH by growing cells in pH 5 and pH 9 conditions. Ultimately, we saw no variation in expression levels in comparison to cells grown in normal LB media. This was the predicted outcome from this experiment as none of the mechanosensitive ion channels in E. coli are known to gate in response to changes in pH. Our data reflects this and supports that the other conditions that we tested truly did affect the expression of each channel.

Now looking at the temperature data on the left, we saw upregulation of MscL and YnaI when grown at 22ºC. YnaI is usually lowly expressed, so this upregulation is another notable finding.
At 22ºC, we saw downregulation of YbdG. At both 22ºC and 45ºC, MscM is downregulated.
Channels such as MscS and MscK seem to be unaffected by changes in temperature as they were expressed at relatively similar levels regardless of temperature.

Finally, looking in the purple box, we see our downshock data. This data showed us that not all of the MscS superfamily members are essential for survival in osmotic downshock. MscL shows the largest decrease in expression of their isotonic levels, as do MscS, YbiO, and YbdG. Based on this information, it is these four channels that play a role in survival of osmotic downshock.

Now that we’ve done these experiments, we have more insight into the specific functions of each of these channels and can now say with more confidence what their specific roles in cell survival may be.
Thank you for taking the time to listen to my poster! Have a great day.

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