Exploring Bacterial Mechanosensation
The Malcolm lab focuses on bacterial mechanosensation. Bacteria utilize mechanosensitive channels to survive osmotic downshock, specifically the mechanosensitive channels of large and small conductance (MscL and MscS respectively). We want to understand how bacteria respond to changes in their environment.
Determining the expression of mechanosensitive channels in E. coli
Based on our work determining the average number of mechanosensitive channels we are determining the expression of the 7 mechanosensitive channels in E. coli. We are culturing E. coli in different growth conditions to determine the differences in expression for MscS, MscL, MscK, McM, YnaI, YbdG, and YbiO. We are utilizing qPCR with ribosomal genes (RpoB and RpoS) as internal standards. Our preliminary research shows that some channels are more dynamic than other channels in different conditions. Stay tuned for more exciting results.
Identifying the genes that correspond to observed mechanosensitive channels in P. graminis
We have shown that there are three unique channels observed in P. Graminis membranes, these correspond to the 7 unique genes found in the P. Graminis genome. We are using qPCR and heterologous expression to determine which genes correspond to the observed channels in the membrane.
Determining the quantity and diversity of mechanosensitive channels in bacterial genomes
In E. coli there are 6 MscS superfamily channels and a single copy of MscL, our work on P. graminis showed that there are also 6 MscS superfamily members and a single copy of MscL. There are several bacterial strains that do not contain a homologue to MscL and others that contain varying numbers of MscS superfamily members. In light of this we set out to determine the number of predicted mechanosensitive genes in bacterial genomes. We utilized BLAST searches and alignments to determine the average number of mechanosensitive genes in bacterial genomes.
Our analysis of more than 150 genomes determined that 67% of genomes encode for a single copy of MscL, there are no bacterial strains that encode for more than one copy. Overall, the average number of MscS superfamily channels is 4± 3, ranging from 0 to 17 unique members in a single bacterial genome. Interestingly, we found that the average number of MscS superfamily members correlates with the bacterial phyla. Check out the full paper for more details.
First observation of mechanosensitive channels in P. graminis
The first publication from the Malcolm lab showed the first observation of mechanosensitive channels in Paraburkholderia graminis membranes. Our bioinformatic analysis identified 6 MscS superfamily members: Pg-MscS-1, Pg-MscS-2, Pg-MscS-3, Pg-MscS-4, Pg-bCNGa, and Pg-bCNGb, as well as a single MscL homologue Pg-MscL. In order to directly observe mechanosensitive channels, we developed methods to create giant spheroplasts that are suitable for patch clamp electrophysiology.
Patch clamp experiments on the native membranes show three distinct channel conductances, two ‘small’ channels and a ‘large’ conductance channel. One of the ‘small’ channels opens at ~2/3 of the tension of the ‘large’ conductance channel, which is very similar to MscS and MscL in E. coli membranes. The other ‘small’ conductance channel opens at the same tension as the ‘large’ channel. Check out the full paper for more details.
If you are an undergraduate at UNF interested in working in the Malcolm Lab, email Dr. Malcolm for an application. If you are looking to post-doc in the Malcolm lab, email Dr. Malcolm for more information (at this time there is not funding for a post-doc).