Phosphatidylcholine (PC) is the most abundant phospholipid in the membranes of the human parasite Leishmania. It is synthesized via two metabolic routes, the de novo pathway that starts with the uptake of choline, and the threefold methylation of phosphatidylethanolamine. Choline was shown to be dispensable for Leishmania; thus, the methylation pathway likely represents the primary route for PC production. Here, we have identified and characterized two phosphatidylethanolamine methyltransferases, LmjPEM1 and LmjPEM2. Both enzymes are expressed in promastigotes as well as in the vertebrate form amastigotes, suggesting that these methyltransferases are important for the development of the parasite throughout its life cycle. These enzymes are maximally expressed during the log phase of growth which correlates with the demand of PC synthesis during cell multiplication. Immunofluorescence studies combined with cell fractionation have shown that both methyltransferases are localized at the endoplasmic reticulum membrane. Heterologous expression in yeast has demonstrated that LmjPEM1 and LmjPEM2 complement the choline auxotrophy phenotype of a yeast double null mutant lacking phosphatidylethanolamine methyltransferase activity. LmjPEM1 catalyzes the first, and to a lesser extent, the second methylation reaction. In contrast, LmjPEM2 has the capacity to add the second and third methyl group onto phosphatidylethanolamine to yield (lyso)PC; it can also add the first methyl group, albeit with very low efficiency. Finally, we have demonstrated using inhibition studies with choline analogs that miltefosine and octadecyltrimethylammonium bromide are potent inhibitors of this metabolic pathway.

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Small colony variants (SCVs) constitute a slow-growing subpopulation with atypical colony morphology and unusual biochemical characteristics that, in the case of clinical isolates, cause latent and recurrent infections. We propose a novel blueprint for the formation of E. coli SCVs through DNA microarray analysis, coupled with complete genome sequencing and verification by qRT-PCR. Our work represents the first proposal for a combination of novel mutations, amplified by a differential shift in expression of select gene groups that work in concert to establish and maintain the SCV phenotype. This combination of genetic and expression events falls under selective pressure, leading to unequal fitness in our strain, SCV IH9, versus its parental strain, BW7261 (a MG1655 descendant). We hypothesize that this combination of events would ordinarily be lethal for bacteria, but instead confers a survival advantage to SCV IH9 due to its slow growth and resistance to acidic and oxidative stress challenges.

As persistent residents of planktonic bacterial cultures, small colony variants (SCVs) constitute a slow-growing subpopulation with atypical colony morphology and unusual biochemical characteristics that, in the case of clinical isolates, cause latent and recurrent infections. We propose a novel blueprint for the formation of E. coli SCVs through DNA microarray analysis, coupled with complete genome sequencing and verification by qRT-PCR. While others have used DNA microarrays to study quorum sensing in E. coli SCVs, our work represents the first proposal for a combination of novel mutations, amplified by a differential shift in expression of select gene groups that work in concert to establish and maintain the SCV phenotype. This combination of genetic and expression events fall under selective pressure, leading to unequal fitness in our strain, SCV IH9 versus its parental strain, BW7261 (a MG1655 descendant). We hypothesize that this combination of events would ordinarily be lethal for bacteria, but instead confers a survival advantage to SCV IH9 due to its slow growth and resistance to acidic and oxidative stress challenges.