Regulation of isoprenoid precursor pathways in Listeria monocytogenes

Listeria monocytogenes is a facultative, Gram-positive intracellular pathogen that is also a model organism for studying bacterial pathogenesis. Much of the appeal of using L. monocytogenes as a model organism derives from the fact that much of the knowledge gained from studying L. monocytogenes pathogenesis or metabolism can be applied to other pathogens that are more difficult to work with or manipulate. However, the unique aspects of L. monocytogenes biology are equally fascinating and still shed light on the host cell processes that help protect against infections./nIsoprenoids are a diverse class of compounds produced by all forms of life and are synthesized from essential precursors derived from either the mevalonate pathway or the nonmevalonate pathway. Most organisms have one pathway or the other, but L. monocytogenes is unique because it encodes all of the genes from both pathways. Other scientists reported that the mevalonate pathway was essential for growth, but here we report that the nonmevalonate pathway was sufficient for growth anaerobically. Deleting the mevalonate pathway gene hmgR (∆hmgR) impaired bacterial growth in all L. monocytogenes strains studied, but the laboratory strain 10403S grew significantly slower than two lineage I strains, FSL N1-017 and HPB2262 Aureli 1997. The faster anaerobic growth of the lineage I strains was initially traced to a difference in the nonmevalonate pathway enzyme GcpE, and then chimeric proteins were constructed to precisely identify the molecular basis of this phenotype. Three amino acid residues, K291T, E293K, and V294A were shown to be necessary and sufficient to increase the anaerobic growth rate of 10403S ∆hmgR. However, these mutations did not map to GcpE in a way that provided any obvious mechanistic insights to how the enzyme function was altered. Even though the nonmevalonate pathway was found to function anaerobically, we were unable to identify conditions where deleting the nonmevalonate pathway impaired L. monocytogenes growth of any strain. Given the fitness cost of maintaining all seven genes in the nonmevalonate pathway, it is likely that there are yet unidentified anaerobic growth conditions that require the nonmevalonate pathway. This work showed that, contrary to previous reports, the mevalonate pathway is not essential for L. monocytogenes growth anaerobically, although it is still essential for growth aerobically./nAfter finding specific mutations in GcpE that altered growth, we were also interested in understanding the broader networks regulating the nonmevalonate pathway in L. monocytogenes. The difference in anaerobic growth rates between 10403S ∆hmgR and lineage I ∆hmgR mutants indicated that it could be possible to find suppressor mutations that increased 10403S ∆hmgR growth rate. 10403S ∆hmgR cultures were passaged anaerobically, and fast-growing suppressor mutants began arising after three passages, which was noted by the observation that cultures initially required four or five days to reach stationary phase, but later only required two days to reach stationary phase. Whole genome sequencing of these cultures identified mutations in several genes, but the most common mutations were in the histidine kinase of a two-component system LisRK. We demonstrated that the suppressor mutations altered rather than disrupted LisRK signaling, but were unable to identify specific genes in the regulon that were responsible for the increased rate of anaerobic growth. This work showed that multiple mutations can be made that alter the rate of L. monocytogenes growth using the nonmevalonate pathway. The previously identified mutations in GcpE likely directly change enzyme kinetics, allowing for increased flux through the nonmevalonate pathway, but the additional mutations identified here likely change gene expression levels or alter metabolite concentrations at points upstream or downstream of the nonmevalonate pathway itself.

Advisor: Portnoy, Daniel A