High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism

Abstract

The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets.

Phyletic distribution and conservation of the bacterial transcription termination factor Rho

“…Since rho inactivation (Griffin et al, 2011), but not BCM treatment (Nishida et al, 1972), inhibits mycobacterial growth, the data also argue that sensitivity to the ATPase inhibitor BCM (Skordalakes et al, 2005) is not always a reliable gauge of the functional significance of Rho.…”

Novel regulatory roles of cAMP receptor proteins in fast-growing environmental mycobacteria

“…The reason why Crp2 appears to be essential for growth of M. smegmatis remains unknown. Analysis of the genes regulated by Crp2 (Table S3) reveals that a homologue of MSMEG_6041 in M. tuberculosis encoding fadE34 is essential for growth on cholesterol (Griffin et al, 2011). Whether MSMEG_6041 is essential for growth of M. smegmatis is not known.…”

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

“…This enzyme shows a very similar homotrimeric fold and catalyses both the dCTP deamination reaction and the triphosphate hydrolysis of the resulting dUTP, directly producing dUMP from dCTP. However, the efficiency of the triphosphate hydrolysis by the bifunctional enzyme is several hundred fold less [19] than that of the monofunctional trimeric dUTPase [20].The monofunctional dUTPase (dut) is essential in Mycobacteria [21][22][23]. In contrast, earlier mutagenesis studies found that the presence of the bifunctional dCTP deaminase:dUTPase enzyme is dispensable for growth in M. tuberculosis [21,22].…”