Membrane proteins constitute 25-30% of the predicted proteins encoded in all genomes so far sequenced. However, even in a well-characterized organism such as Escherichia coli, the functions of nearly half of the predicted membrane proteins are only poorly understood. By the use of genetics to isolate novel mutants, the laboratory has acquired the tools to understand the roles of a previously uncharacterized but widely distributed and conserved gene family in lipid biosynthesis and cell division, the dedA family. DedA is a large family of inner membrane proteins found widespread in eubacteria and some Archaea. Genes for these proteins are also present in the genomes of certain green algae but have not been identified in other eukaryotic genomes. Currently, there are more than 1000 genes in the online database annotated as being dedA family members or possessing significant amino acid identity to E. coli DedA. To date, no function has been assigned to the DedA protein family. This is due largely to the inability to study the proteins genetically, as each single deletion mutant that has been made in E. coli is without an observable phenotype. A new temperature sensitive mutant, BC202, with alterations in membrane phospholipid composition and defects in cell division was isolated. BC202 has targeted deletions of two related dedA family genes (yqjA and yghB) and growth is restored by expression of either of these genes. YqjA and YghB have 61% amino acid identity so the proteins are expected to have redundant functions. BC202 is a powerful tool to study the roles of lipids in bacterial cell division and the functions of the conserved DedA superfamily of membrane proteins. The specific aims of this project are (1) to determine the lipid and cell division defects in the yghB, ygiA double mutant; (2) to determine the function(s) of YghB and YqjA; and (3) to determine the functional conservation of DedA family members from divergent bacterial species.
Broader Impacts Results from the project will lead to a deeper understanding of the roles of lipids in both prokaryotic and eukaryotic cells. In an effort to enhance scientific and technological understanding, the results from these studies will be promptly published in peer-reviewed scientific journals. Materials obtained, including cloned genes and bacterial mutants will be made available for broad dissemination free of charge. The PI is keenly aware of the need to promote teaching and training of graduate and undergraduate students and will train students in the research laboratory and also will integrate the research in instruction by including aspects of the project in his teaching assignment in Microbial Physiology. The PI is also committed to broadening participation to include members from underrepresented groups in biology in his research. The PI maintains an ethnically diverse laboratory and coordinates collaborations with faculty and student researchers from Louisiana State University and Southern University.
We began this project with the goal of identifying the functions of an unknown protein family named the "DedA family". Proteins belonging to this family are embedded within the cell membrane and therefore are termed membrane proteins. The functions of many membrane protein families are still poorly defined. We now know that this family is required for drug resistance in Escherichia coli and therefore will be of great interest to the biomedical community. One reason we were interested in the DedA family is that it is present in virtually every bacterial genome so far sequenced. This level on conservation indicates that members of the family carry out a basic function important to every cell. The tool we used in our investigations was an E. coli mutant we discovered with deletions of TWO genes (named yqjA and yghB). It turns out that the YghB and YqjA proteins (belonging to the larger DedA family) are very similar to each other in terms of amino acid sequence, sharing more that 60% of their amino acids. This high degree of similarity means that these two proteins can substitute for each other, so if you delete either gene alone the cell is fine, and grows normally. However, if you isolate a strain with deletions of BOTH genes the cells are VERY sick (but viable). They cannot complete the process of cell division (Figure 1) and cannot grow at high temperatures (> 42 °C, a temperature at which wild type E. coli grows well). These observations indicated that these genes are doing something very important for the cell. (We have named this strain BC202 after a former technician who isolated it). We showed conclusively that the problem with cell division is caused by the inability of BC202 to secrete enzymes called amidases across the cell membrane. Amidases are involved in remodeling the bacterial peptidoglycan cell wall and are required for the cell to complete cell division. The problem with BC202 is not the amidases themselves, but mechanism by which they are secreted. This secretion system is called the TAT or "twin arginine transport" pathway. The TAT pathway in turn requires the proton gradient across the membrane a.k.a. the protonmotive force (PMF) to operate efficiently. It is this PMF that is not maintained in BC202. In fact, we can rescue the growth and cell division defects of BC202 simply by restoring a component of the PMF (the pH gradient) by growing the cells at a slightly acidic pH of 6.0. BC202 is also not viable at a slightly basic pH of 8.0. These results imply that a probable function of YqjA/YghB is to import protons into the cytoplasm. Most proton importers use a mechanism where they exchange the proton for another ion of like charge such as sodium or potassium. These types of transporters are called antiporters. In fact, one very well understood family of proton antiporter- the major facilitator superfamily (MFS)- is also capable of exchanging protons for drugs and biocides and therefore capable of providing drug resistance to many species of bacteria by "pumping" the drug out of the cell. Importantly, the DedA family displays absolutely no amino acid identity to the MFS family. However, due to our cumulative results suggesting that the function of the DedA family is to import protons into the cell, we tested whether BC202 was sensitive to any commonly used antibiotics and biocides that are known to be substrates of these above mentioned drug pumps. While BC202 is not sensitive to all antibiotics, it is extremely sensitive to a number of commonly used biocides. Indeed, BC202 is up to ten times MORE sensitive to certain compounds than mutants missing members of the well characterized MFS family. Our work analyzing a novel mutant strain has identified a previously unidentified drug transporter family of proteins that contribute to drug resistance of E. coli and likely many species of bacteria. Figure 1. BC202, with deletions of yqjA and yghB, cannot complete cell division forming chains at the permissive growth temperature of 30 °C. Normal cell division (left panel) proceeds by a carefully orchestrated process involving constriction at the cell center and separation of daughter cells (left panel). The arrows in the right panel show the locations of sites where division is begun but blocked in BC202 due to loss of proper secretion of periplasmic amidases. Figure 2. YqjA transports protons into the cell coupled with symport or antiport of an uncharacterized substrate. We now believe YqjA/YghB is capable of exporting drugs and other toxic compounds based upon the sensitivities of the mutant BC202.
