Submitted by Kathleen Ryan on
Gram-negative bacteria are enclosed in a three-layer envelope, composed of the inner or cytoplasmic membrane (IM), a thin layer of peptidoglycan (PG), and an outer membrane (OM). The OM is an asymmetric bilayer, with phospholipids populating the inner leaflet and lipopolysaccharide (LPS) predominating in the outer leaflet. LPS is a glycosylated lipid that is anchored in the outer leaflet of the OM by its lipid A segment (Whitfield and Trent, 2014). LPS confers robust barrier function upon the OM, making it inherently less permeable than the IM to small hydrophobic compounds, including many antibiotics (Nikaido, 2003). It is widely accepted that the lipid A portion of LPS is strictly required for the viability of Gram-negative bacteria.
Surprisingly, we recently demonstrated that the Gram-negative bacterium Caulobacter crescentus is capable of surviving without lipid A (Zik et al., 2022). Survival of lipid A-deficient Caulobacter is dependent upon two conditions: 1) inactivation of the iron-responsive transcriptional regulatory protein Fur and 2) the presence of a novel anionic sphingolipid, ceramide phosphoglycerate (CPG). Caulobacter is only the fourth Gram-negative bacterium shown to survive when its lipid A is genetically eliminated. Due to the scarcity of lipid A-deficient strains, both the mechanisms that support viability in the absence of lipid A and the consequences for OM structure and function are almost completely unknown. In this project, Caulobacter strains deficient in either lipid A or CPG will be used to dissect the contributions of different lipids to important cellular phenotypes that depend on the OM, such as resistance to antibiotics, osmotic stress, starvation, and other stresses.
For background information about LPS, lipid A, and the Gram-negative outer membrane, check out
doi: 10.1146/annurev-biochem-060713-035600 (Whitfield and Trent, 2014) and doi: 10.1128/MMBR.67.4.593-656.2003 (Nikaido, 2003).
For our recent paper on the discovery of lipid A-deficient Caulobacter crescentus, see doi: 10.1016/j.celrep.2022.110888 (Zik et al., 2022).
Undergraduates will be learning standard techniques in microbiology in order to characterize the resistance or sensitivity of wild-type and lipid A-deficient Caulobacter strains to a variety of stresses. You will measure bacterial growth and survival and learn to use light microscopy to observe and document alterations in cell shape. Duties will also include the preparation of culture media, chemical solutions, and sterile equipment used directly in experiments. Undergraduates are expected to attend and participate in weekly lab meetings to discuss experimental results and the relevant scientific literature. Undergraduates will be expected to work as a team on some experiments. To make progress on this project, you should expect to spend about 10 hours/week in the lab.
Undergraduates who apply should have taken Chem 1, AP Chemistry, or the equivalent course at another college (should know how to make solutions of specified concentrations using solid chemicals). Must be well organized, reliable, and self-motivated. Should be able to communicate clearly and in a timely manner with the PI and undergraduate colleagues in the lab. Above all, please be excited about microbiology and the scientific method!