In this work, we have significantly refined our understanding of the cellular functions necessary for the viability of Mtb. While others have used similar deep sequencing methods to map insertion sites in other organisms 
our new analytical tools allowed the statistically rigorous prediction of the genes essential for the viability of Mtb. The majority of these essential genes are consistent with those found by previous microarray-based methods. However, significant differences in these predictions were also noted, and the majority of these are attributable to technical and analytical refinements. As a result, this work provides a much more precise and statistically rigorous global assessment of essentiality than previously possible.
A few genes that we found to contain very significant gaps in transposon coverage have been successfully deleted, and produce strains that grow normally under similar conditions to those used in our study 
. The reasons for these apparently contradictory results likely vary for each gene. It is possible that some of these genes are truly dispensable for in vitro
growth, and the identified gaps in transposon coverage are either due to unappreciated transposon specificity, or selection against specific protein truncations. Conversely, it is possible that deletion mutants lacking these genes appear to grow normally because extragenic suppressor mutations have accumulated. This phenomenon is well-documented for mutants generated by homologous recombination 
, but is very unlikely to affect transposon-mapping studies. These differences highlight the advantages and limitations of different genetic approaches for defining essential genes.
In addition to this qualitative analysis, we also quantitatively compared mutant pools grown under different conditions to understand how Mtb metabolizes cholesterol. A number of recent studies have demonstrated that Mtb mutants lacking the capacity to acquire or degrade cholesterol are defective for growth in animal models of TB 
. In order to define a discrete set of ORFs required for growth on this carbon source, we applied a continuous function cutoff to our comparative data, which placed equal importance on fold change in representation and statistical significance. More traditional one-dimensional cutoffs would have excluded genes that were only moderately underrepresented but exceptionally significant, which our analysis includes. Nonetheless, in order to avoid false positive predictions, we set a relatively stringent cutoff, and it is likely that even more than the 96 identified genes contribute to growth in cholesterol.
In addition to the dedicated cholesterol catabolic functions, we found that the use of this compound as a source of both cellular energy and biosynthetic carbon requires a variety of central metabolic pathways. Some of these requirements are likely due to the simple shift from a glycolytic substrate to one that relies heavily on β-oxidation. However, the precise pathways we identified appear specific to the mixture of metabolites derived from cholesterol. For example, gluconeogenesis under these conditions is likely to be initiated by the conversion of pyruvate to PEP via the action of pyruvate phosphate dikinase (PpdK), whereas this pathway relies exclusively on phosphenolpyruvate carboxykinase (PckA) during growth on acetate 
. In other bacteria, PpdK-mediated gluconeogensis is favored during growth in the presence of pyruvate 
. As pyruvate is produced both as a direct product of sterol catabolism and through the activity of the methylcitrate cycle, we speculate that the relative abundance of cholesterol-derived pyruvate favors the PpdK-mediated pathway. These observations indicate that different gluconeogenic pathways may be preferentially used by Mtb depending on the relative abundance of precursor metabolites.
Cholesterol acquisition is predominantly required for bacterial persistence during the chronic stage of Mtb infection in mice and for growth in the cytokine-stimulated macrophages that characterize this stage of infection 
. While cholesterol is metabolized by the bacterium throughout infection 
, we have shown that this ability is not required for the initial growth of the pathogen in acutely-infected animals or in the resting macrophages that model this early pre-immune period. A recent study demonstrating that cholesterol catabolism is not necessary for bacterial growth in acutely-infected guinea pigs or a macrophage cell line confirms these observations 
. Thus, it appears that a mixture of carbon sources, including cholesterol, fuel the initial growth of the bacterium, and this sterol becomes a uniquely essential nutrient in chronically-infected animals. Based on these observations, we predict that the requirements for both the dedicated sterol catabolic enzymes, as well as the central metabolic pathways we have defined, are likely to change as infection proceeds.
Identifying the full complement of cellular functions necessary for cholesterol utilization has also revealed the scale of this nutritional adaptation during infection. When we compared these data to previous genome-wide screens for mutants attenuated in mouse models of infection 
we found that a full ten percent of genes specifically required for bacterial growth in vivo
are also required for the utilization of cholesterol in vitro
). These genes encode both dedicated sterol catabolic functions, as well as enzymes involved in central metabolism. Thus, while it is clear that Mtb must adapt to a variety of host-specific conditions to sustain a productive infection, our data suggest that a single nutritional change is responsible for a significant portion of this adaptation. These cholesterol catabolic functions, in conjunction with the hundreds of other genes that we found to be essential for bacterial viability, both expand and refine the repertoire of targets for new TB therapies.
Whole genome profiling techniques have proven to be useful tools for understanding complex pathways, such as those required for cholesterol utilization. Most of these approaches rely on determining the relative transcript or protein abundance. However, these strategies make a major assumption – that critical genes are tightly regulated in response to metabolic changes. While this may be true in many cases, it is often not. In order to avoid this assumption, we have directly identified the genes required for growth. While every approach has its own inherent strengths and weaknesses, the phenotypic profiling strategy described here is a powerful complementary tool for understanding bacterial physiology.