Neisseria meningitidis is Gram-negative bacteria that can causes acute bacterial meningitidis and life-threatening sepsis. Pili of N. meningitidis are a major virulence factor and are subject to several different post-translational modifications. Among these modifications, the presence of phosphorylcholine (ChoP) and a o-linked glycan are both phase-variable (subject to high frequency, reversible on/off switching of expression). Previously, we showed that both pili-linked ChoP and glycosylation of N. meningitidis could mediate meningococcal adherence to platelet activating factor receptor on human airway cells. However, N. meningitidis does not contain genes associated with previously described ChoP synthesis pathways found in other bacteria, and it does not require phosphorylcholine from growing media for incorporation onto pili. Our previous studies have demonstrated that ChoP modification was increased when N. meningitidis were grown in a chemically defined choline free medium (CDM) compared to the rich medium, and differently expressed genes between BHI culture and CDM culture are determined by RNA-Seq. In this study, we aim to identify differently expressed genes that may be potentially involved in the biosynthesis of ChoP and characterize the novel, N. meningitidis ChoP biosynthetic pathway as the adjunct to understanding its role in pathogenesis.