Sequencing transposon mutant libraries have been pivotal in annotating essential and non-essential genes in bacteria. This is particularly very helpful in the case of Mycobacterium tuberculosis with a large part of its genome without known function. It is not known whether there are any variations in the essentiality states as a function of optimal growth in the absence of any selection pressure. We here grow a high-density mutant library of M. tuberculosis through serial cultures and monitor the temporal fluctuations in insertion frequencies across all TA dinucleotides in the genome. Genes that cause morphological and physiological heterogeneity or enable metabolic bypass were found to gradually lose insertions, while genes comprising the toxin-antitoxin systems were found to get enriched with insertions during growth in nutrient replete conditions. High levels of fluctuations were observed in genes involved in cell wall and cell processes, intermediary metabolism, and genes involved in virulence, suggesting new modes of adaptation undertaken by the mutants. We also report the essentiality status of several newly annotated genetic features.Copyright © 2020 Elsevier B.V. All rights reserved.